Robert N. Moore

Dynorphin and related opioid peptides enhance tumoricidal activity mediated by murine peritoneal macrophages.

The influence of dynorphin A (DYN) and related opioid peptides on the tumoricidal function of activated murine peritoneal exudate macrophages (PEM) was investigated. Addition of DYN to macrophage cultures previously activated with mixed α + β‐inter‐feron (IFN‐α/β) and bacterial lipopolysaccharide (LPS) significantly enhanced their ability to lyse P815 murine mastocytoma cells in a 16 hr chromium‐release assay. The effects of DYN were dependent on prior macrophage activation. Peptide subfragments of DYN were effective in a manner similar to that of the 17‐amino‐acid parent molecule, indicating that peptide interaction with either κ or δ‐opioid receptors on the effector cell is effective in potentiating lytic function. The involvement of opiate receptors was confirmed by inhibition of the effects of DYN and leucine enkephalin by the opioid receptor antagonist naloxone. Finally, in addition to IFN‐α/β‐primed macrophages, DYN also augmented tumoricidal function in PEM primed for cytotoxicity by either γ‐interferon (IFN‐γ) or the calcium ionophore A23187, Indicating that DYN potentiates function in activated macrophages independent of the specific mode of activation.

Interferon produced endogenously in response to CSF-1 augments the functional differentiation of progeny macrophages.

The role of endogenously produced interferon α/β in the functional maturation of newly derived mononuclear phagocytes was investigated. Addition of highly specific anti‐interferon α+ βantiserum to murine marrow cultures stimulated with colony‐stimulating factor‐1 (macrophage growth factor) markedly suppressed the capacity of resulting progeny mononuclear phagocytes to ingest opsonized sheep erythrocytes (EAlgG). This impairment was corrected either by direct addition of interferon α+ βat a concentration in excess of that neutralized by the antiserum or by the addition of lesser amounts of interferon (33 U/ml) following removal of the anti‐interferon from the cultures. Conditioned media from control colony‐stimulating factor‐stimulated cultures similarly reversed the impairment of maturation resulting from 5 days of growth in the presence of anti‐interferon. This enhancement of EA|gG ingestion reflected upon the interferon activity in the conditioned media and was neutralized by anti‐interferon. Lastly, the endogenous interferon was found to enhance EAlgG ingestion by a majority of the mononuclear phagocyte progeny and not by a limited subpopulation.

Differential effects of CD4+ and CD8+ cells in acute, systemic murine candidosis

Monoclonal antibody (mAb) depletion was used to assess contributions of CD4+ and CD8+ cells in resistance to systemic murine Candida albicans infection. Depletion of CD8+ cells did not influence either survival or mean survival time (MST); however, depletion of CD4+ cells significantly enhanced both survival and MST. Combined depletion of both CD4+ and CD8+ cells significantly lengthened the MST but did not enhance survival. A protective influence of CD8+ cells could be deduced but, to be manifested, required depletion of an overshadowing immunopathologic CD4+ response.

Calcium ion involvement in the action of Pasteurella haemolytica leukotoxin

The influence of Ca2+ ions on the cytotoxic activity of Pasteurella haemolytica leukotoxin was investigated. The divalent cation influenced the cytotoxic effect of the leukotoxin for sensitive BL-3 target cells, but its absence did not eliminate cytotoxicity. In short-term 1-h assays using neutral red uptake as a measure of cell viability, depletion of Ca2+ either by exhaustive dialysis or by addition of the Ca2+ chelators EDTA and EGTA eliminated the cytolytic effect of low doses of the toxin. Addition of Ca2+ to target cell cultures depleted of the divalent cation restored the cytolytic effect of the leukotoxin. Prolonged exposure of the BL-3 cells to the toxin abrogated the protective effect of EDTA and EGTA. Cell death measured by uptake of neutral red, exclusion of trypan blue and 51Cr release indicated that protection observed in the absence of free Ca2+ was temporary. Toxin-induced cytolysis equivalent to that observed in the presence of Ca2+ occurred following the initial 2-h exposure. In addition, verapamil, a Ca2+ channel blocker, prevented cell death during 1-h cytotoxicity assays. The protection afforded by verapamil was dose-dependent and was influenced by the concentration of Ca2+ in the buffer medium. The results suggest that Ca2+ positively influences the rapid initial phase of cell death resulting from exposure to the toxin, but is not required for the entirety of the cytolytic process.

Bordetella bronchiseptica fimbrial protein-enhanced immunogenicity of a Mannheimia haemolytica leukotoxin fragment.

Leukotoxin produced by Mannheimia (Pasteurella) haemolytica is an important virulence factor in shipping fever pneumonia in feedlot cattle and is a critical protective antigen. In this study, the immune response to a chimeric protein generated by combining a gene fragment encoding neutralizing epitopes of M. haemolytica leukotoxin and a fimbrial protein gene (fim N) from Bordetella bronchiseptica was evaluated. The recombinant gene was cloned in a bacterial expression vector under the control of the tac promoter and expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Immunization of mice with the recombinant protein, GST-LTXFIM elicited a significantly stronger anti-leukotoxin antibody response than comparable immunizations with GST-LTX fusion proteins lacking FIM N. The GST-LTXFIM was also more stable than GST-LTX during storage at -80 degrees C, thus alleviating a stability problem inherent to leukotoxin. This chimeric protein may be a candidate for inclusion in new generation vaccines against shipping fever pneumonia.

Isolation and functional studies on feline bone marrow derived macrophages

Abstract In this report, we describe an in vitro culture method for feline bone marrow cells, which yields large numbers of quiescent macrophages after 14 days of culture. The bulk of the cultured cell population consists of macrophages as assessed by morphology, macrophage specific cytochemistry, and phagocytosis. The remaining cells were lymphocytes, bone marrow stromal cells, fibroblasts and occasional polymorphonuclear leukocytes. While resting cells produced no detectable interleukin 1, stimulation with lipopolysaccharide (LPS) induced the production of biologically active interleukin 1. After 6 h LPS stimulation, mRNA for tumor necrosis factor α and interleukin 1β was detectable. The absence of mRNA in unstimulated cells indicates cultured macrophages were not activated until stimulated by LPS or plastic adherence. This approach provides a useful means to measure potential modulatory effects by virus infections or other agents upon feline macrophage gene expression.

Regulation of macrophage accessory functions by interactions involving lymphokines and endotoxin

SummaryIt is important to reemphasize the observation that two distinct regulatory molecules, colony-stimulating factor (CSF) and interferon, can directly influence macrophage accessory functions. CSF stimulates macrophages to secrete interleukin 1 (IL1) whereas interferon induces a conversion to Ia antigen expression. Either directly or indirectly these functions can also be influenced by a combination of lymphokines and bacterial lipopolysaccharides (LPS). As is the case with effector functions, it is apparent that specific regulatory molecules exist which function to amplify the influence of microbial products on macrophage accessory activities.

Mediator interactions regulating macrophage secretion of interleukin 1 and interferon.

The mononuclear phagocyte system plays a critical role in host resistance to infection. The capacity of mononuclear phagocytes to deal with microorganisms appears, however, to be under regulatory control rather than static. Two mediators which appear to act in the process of macrophage differentiation and activation are interferon and colony-stimulating factor(s) (CSF). A substantial body of evidence indicates that interferon stimulates a variety of macrophage functions including phagocytosis, spreading, killing of microorganisms and tumor cells (1) and, recently, la-antigen expression (2). The effects of CSF are, however, less well known. The purpose of the present communication is to discuss recent findings which indicate that CSF as well as interferon can participate in the process of macrophage activation.

Endocrinelike Activities of the RES

Numerous findings during the past decade have identified an impressive secretory capacity of the phagocytic cells of the reticuloendothelial system (RES). Macrophages in vitro have been found to secrete a variety of potent hormonelike mediators that influence or regulate activities of a variety of other cell types as well as other cellular components of the RES. In many cases, a direct correlation can be made between in vitro findings and established events occurring in vivo during infection or inflammatory responses, reaffirming the importance of the RES in a variety of host-microbial interactions. Of necessity, these mediators have been defined by their in vitro functional activities. As a result, there has been an accumulation of seemingly unrelated data concerning a dramatic number of different mediators. Recent studies, however, indicate that the actual number of different mediators produced by monocytes and macrophages is more limited than that suggested by the available literature. Investigations designed to characterize and to isolate specific mediators have shown that several are in fact the same molecules with multiple functional activities.

Nanomolar cyclic adenosine and guanosine monophosphates stimulate macrophage colony-stimulating factor responsiveness by murine transitional progenitors.

Both 3:5′ cyclic adenosine monophosphate (cAMP) and 3′:5′ cyclic guanosine monophosphate (cGMP) stimulated colony‐stimulating factor 1 (CSF‐1)–dependent colony formation by murine two‐signal–dependent progenitors without influencing colony formation by committed CSF‐1–responsive progenitors. The stimulatory effect was optimal at 10‐9 M and did not diminish with increasing concentrations of the cyclic nucleotides. The membrane‐permeating analogs dibutyryl cAMP and 8‐Br‐cGMP similarly augmented colony formation by the transitional progenitors at 10‐9 M; however, with increasing concentration, enhancement diminished with eventual inhibition of total colony formation at micromolar concentrations. Stimulation by the two cyclic nucleotides was mutually incompatible. The results indicate that physiological levels of extracellular cyclic nucleotides may significantly influence myelopoiesis. Furthermore, the results introduce the interesting possibility that stimulation, unlike inhibition, may be initiated through an extracytoplasmic mechanism that does not require direct activation of cytoplasmic cyclic nucleotide–dependent protein kinases.